Light activation of the hydrolysis of cyclic GMP is hypothesized to transiently decrease the dark current in rod photoreceptor cells. Termination of this hydrolysis and synthesis of cyclic GMP to restore its concentration are thought to underlie the recovery of the dark current. While the biochemical reactions involved in the activation phase are understood in great detail, there are many unresolved questions about the kinetics and regulation of the reactions in the recovery phase. Guanylate cyclase, the enzyme that synthesizes cyclic GMP from GTP, plays a critical role in the recovery process. In continuing our studies on this enzyme, the proposed research will utilize biochemical, molecular biological, immunohistochemical and electron microscopic techniques to address the following questions: (1) Are there multiple cyclases in the rod photoreceptor cells? Where in the cell are they localized? (2) Are the effects of recovering and ATP on cyclase mediated by other proteins? (3) Are the cyclases regulated by phosphorylation and dephosphorylation? (4) Are the cyclases modulated by the reaction product, pyrophosphate, either directly or through a calcium mediated process involving pyrophosphatase? (5) Are the cyclases activated by light? Are the proteins of the activation phase of light response, such as PDE, G-protein, arrestin, coupled to cyclase in mediating the effects of light? The proposed research will enhance our understanding of the controls on cyclic GMP synthesis and thus on the recovery phase of the light response.